DNA-based transactions in the eukaryotic nucleus, such as transcriptional activation and repression, replication, rearrangements, and repair, occur in the context of chromatin. Long commonly thought to be an inert packaging scaffold, it is increasingly evident that signaling to nucleosomal histones is a controlling feature of genomic processes. One key class of histone alterations is post-translational modification (PTM), largely of their amino and carboxyl termini. Detailed evidence initially emerged for acetylation (ac), however histones are also phosphorylated (ph), methylated (me), ubiquitylated (ub), and sumoylated (su). The scale and intricacy of regulation via histone PTMs (hPTM) is only beginning to be appreciated and there are more complex layers of regulation via hPTMs yet to be elucidated. One key question regarding histone hPTMs is whether there is a predictive relationship linking individual PTMs and specific genomic processes. hPTMs are centrally important to regulate binding of key effector proteins. Our hypothesis is that regulatory information and effector protein binding is signaled by combinatorial hPTM patterns overtime and in 3- dimensional space. We will investigate several classes of hPTM cross-talk involved in transcriptional regulation in S. cerevisiae. The first is a temporal sequence of H2B ubiquitylation followed by its deubiquitylation during gene activation. The second is an alternative pattern of repressive H2B sumoylation vs activating histone acetylation/ubiquitylation. The third is a combinatorial pattern consisting of S10pi/K14ac on H3 during transcriptional initiation. Our specific goals in the proposed research are (1) to investigate mechanisms underlying the contrasting roles of histone ubiquitylation in transcriptional activation, compared to histone sumoylation in repression. Binding proteins to H2Bub and H2Bsu will be identified, and examine how these PTMs dictate chromatin structure. (2) To determine mechanisms involved in Ubp8-dependent deubiquitylation of H2B during transcriptional activation. We will test a model from our previous results, proposing that H2Bub functions as a barrier to phosphorylation on RNA polymerase II, and that a SAGA related complex containing Ubp8 proteolyzes ubiquitin from H2B within the ORF to promote transcript elongation. (3) To examine how the combinatorial pattern H3S10pi/K14ac regulates transcriptional activation. We believe that histone phosphorylation is a nexus to both inhibit binding of negative corepressors of transcription and, within the S10pi/K14ac pattern, promote binding of positive coactivators. It is now also known that aberrant histone PTMs underlie human disease. Indeed, histone deacetylase inhibitors constitute a promising class of anticancer medicines, and are already utilized in humans. Hence, elucidation of mechanisms and physiological roles of complex hPTMs may significantly impact our understanding and ultimate treatment of disease.